The objectives of this research program are to establish the mechanisms by which gallbladder ganglion neurons and smooth muscle cells function to promote proper filing and emptying the of the organ, and how these functions are altered in disease. Approximately 20 million people in the USA have gallstones or have had a cholecystectomy; the associated annual direct costs are comparable to the entire NIH budget. The proposed projects are designed to test hypothesis related to the cellular and subcellular mechanisms associated with disease-related hypomotility of the gallbladder, and to identify the neurotransmitters that mediate gallbladder neuromuscular transmission. The first specific aim is to determine the cellular mechanisms by which elevated cholesterol in the bile decreases gallbladder contractility. These experiments include evaluation of cholesterol's effects on ligand activation of receptors, signal transduction, channel function, and intracellular calcium mobilization. This aim will involve laser scanning confocal calcium imaging, patch clamp and intracellular recording from gallbladder neurons to test the hypotheses that cholesterol alters the ionic properties of smooth muscle and the responsiveness of gallbladder neurons and muscle cells to investigate whether opiates and somatostatin elicit biliary stasis by suppressing neuromuscular transmission in ganglia and/or at the smooth muscle. We will test the hypothesis that these compounds decrease gallbladder motility by acting presynaptically to decrease the release of excitatory transmitters in ganglia and at the neuromuscular function. The third specific aim is to identify mediators of neuromuscular transmission in the gallbladder. We use intracellular recording form muscle cells, and measurement of muscle tension, while stimulating nerves of the gallbladder to test the hypothesis that neuromuscular output from gallbladder ganglia is entirely excitatory, consisting of cholinergic and tachykinin release from local neurons onto the muscle. The proposed project will examine gallbladder function the tissue to subcellular levels, from neurons to smooth muscle, and from ensemble to single channel activity. Any array of techniques will be used, including intracellular recording, global and subcellular calcium imaging, radioimmunoassay, immunohistochemistry, and whole cell and single channel patch clamping. In this way, we hope to provide an unique, integrated view of gallbladder neuromuscular function in health and disease.